Metalloproteinases comprise a family of matrix degrading enzymes that are believed to play a role in normal development as well as in tissue remodeling and repair. Abnormal expression of metalloproteinases may also contribute to the pathogenesis of many destructive processes. We have recently cloned a murine macrophage elastase (MME), that is a distinct member of the metalloproteinase gene family with potent elastolytic activity. Subsequently, we cloned and expressed the human orthologue to MME that we call human macrophage metalloelastase (HME). Deduced amino acid sequence demonstrates that HME is a unique human metalloproteinase. HME mRNA and protein are expressed in human alveolar macrophages derived from several smokers. Similar to MME, both native and recombinant HME degrade elastin. The long-term goals of this grant are to test the central hypothesis that macrophage elastase contributes to the pathogenesis of destructive inflammatory lung diseases. Particularly, we will focus on the role of macrophage elastase in pulmonary emphysema related to cigarette smoking which is characterized by macrophage accumulation and elastin degradation. To achieve these goals, we will first define the elastolytic and other proteolytic properties of human and murine macrophage elastase. Recombinant macrophage elastase will be used to determine the binding affinity and kinetic parameters of catalysis for elastin. We will also delineate the capacity of macrophage elastase to degrade other relevant extracellular matrix molecules as well as alpha-1-antiproteinase. Cleavage sites will be determined on selected susceptible matrices to begin to characterize peptide bonds cleaved by HME/MME. Second, we will determine the structural basis of the unusual C-terminal processing of macrophage elastase and investigate the effect of C-terminal processing on elastin binding, catalysis, and TIMP inhibition. We will identify the C-terminal cleavage site(s), mutate it to prevent the processing event, and then compare the ability of the truncated vs mutant forms to degrade elastin. Third, to address the role of MME in emphysema and other destructive inflammatory diseases we will determine whether HME production by human alveolar macrophages correlates with smoking and emphysema. We will identify mediators of inflammation that regulate HME biosynthesis in macrophages with a future goal being to define the molecular mechanisms responsible for regulation. Fourth, to more directly determine the role of macrophage elastase in emphysema, we will create a strain of mice with a targeted mutation in the macrophage elastase gene and examine the capacity of these MME-deficient mice and normal littermates to develop emphysema and other inflammatory lung diseases.